The goal of this project is to understand the molecular mechanisms governing post-initiation events in postreplicative gene transcription during vaccinia virus infection. In recent years it has become increasingly clear that post-initiation events in transcription, including transcription elongation, termination and RNA cleavage, comprise important control points for regulation of gene expression in both prokaryotes and eukaryotes. Our recent work suggests that postreplicative mRNA 3' end formation during vaccinia virus infection is a concerted process that involves both transcription termination and RNA cleavage, that it is coupled to viral DNA replication, and that it is mediated by both viral and host factors in a dynamic complex. The factors include a viral RNA release factor (A18), a viral RNA cleavage/transcription/DNA replication factor (H5), two viral positive transcription elongation factors (G2 and J3), and at least two host factors required for A18 RNA release activity and for RNA cleavage. This project comprises two aims, focusing on understanding the mechanism of transcription termination and endoribonucleolytic RNA cleavage. Studies of termination (Aim 1) involve mechanistic studies on A18 and its required host factor, and studies of mRNA cleavage (Aim 2) involve mechanistic studies of an endoribonuclease comprised of the viral H5 protein and host proteins. The viral gene products under study are highly conserved among vertebrate poxviruses, they are essential for virus replication, they all unquestionably collaborate in mediating postreplicative gene transcription elongation and termination, and yet their precise roles in these processes are only partially understood. The research proposed here will significantly refine our understanding of the roles of these essential genes in poxvirus replication, and it will also impact on our understanding of the interaction between the virus and the host cell. The results will provide critical insight into fundamental mechanisms of vaccinia virus gene expression in particular, and the system may prove to be an important model for study of regulation of transcription elongation in eukaryotes in general.